Pharmacologic Method Of Lowering Cholesterol Production

ABSTRACT

The present invention provides a screening method for identifying test agents that modulate cell membrane cholesterol activity. The modulating activity of the test agents may be measured using lytic compounds, which cause cell lysis or increases in cell permeability in response to cell membrane cholesterol levels. The invention further provides therapeutic agents that are identified using the screening method. The therapeutic agents either increase or decrease the cell membrane cholesterol activity, cholesterol concentration and/or both in vivo and/or in vitro.

CLAIM OF PRIORITY

This application claims priority from U.S. Provisional PatentApplication No. 60/561,585, filed Apr. 13, 2004, the entire teachings ofwhich are incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under grant No. HL 28448awarded by the National Institutes of Heath. The Government has certainrights in this invention.

FIELD OF INVENTION

The present invention relates to methods and compounds that modulatecholesterol levels as well as screening methods used to identify suchcompounds.

BACKGROUND OF THE INVENTION

Cholesterol is an essential constituent of all animal cell membranes.Cholesterol reduces the permeability of the membrane, increases themembrane's mechanical strength and helps to organize the membraneconstituents laterally into domains. In the cell, cholesterol'sabundance is tightly controlled through biosynthetic, storage,ingestion, and transfer pathways. Through these pathways, cells have theability to adjust their cholesterol level to their needs.

Unfortunately, in some instances, in vivo cholesterol levels are notproperly adjusted. Cholesterol disorders, specifically high serum levelsof cholesterol and the mishandling of cholesterol in cells of arterialwalls, may cause disease and death in humans by contributing to theformation of atherosclerotic plaques in arteries throughout the body. Infact, cholesterol disorders in the United States contribute to such ahigh number of health issues that the National Heart, Lung, and BloodInstitute launched the National Cholesterol Education Program in 1985.The goal of the National Cholesterol Education Program is to contributeto reducing illness and death from coronary heart disease in the UnitedStates by reducing the percent of Americans with high blood cholesterol.The program plans to do this by raising awareness of the link betweenhigh cholesterol levels and coronary heart disease.

Many of the illnesses triggered by cholesterol abnormalities areaddressed through both increased education and drug treatment. Currentdrugs used for treatment of cholesterol related disorders are compoundscalled statins, which inhibit cholesterol biosynthesis by blocking theproduction of the cholesterol precursor, mevalonic acid. However,mevalonic acid is used by the body to synthesize many other importantcellular constituents. Unfortunately, because of the numerous cellularuses for mevalonic acid, statins, which are largely non-specific, oftenhave side effects because they suppress a variety of metabolic functionsother than cholesterol biosynthesis.

Accordingly, there is need to find other therapeutic agents to combatcholesterol disorders as well as methods for identifying such compounds.

SUMMARY OF THE INVENTION

One embodiment of the present invention provides a method of screeningcompounds for cholesterol modulating activity. The screening methodinvolves contacting one or more test agents with one or more cells anddetermining or measuring whether the one or more test agents has aneffect on cholesterol activity, cholesterol concentration or both in amembrane of the one or more cells. The screening method can also involvecontacting the one or more cells with a lytic compound, so that thelytic compound causes perforation or lysis of the membrane of the one ormore cells when the cholesterol activity, cholesterol concentration orboth of the membrane of the one or more cells reaches a level at orabove a threshold cholesterol level. In certain screening methods, thecholesterol content of the membrane can be increased or decreased bycontacting the one or more cells with a cholesterol modulating compound.

In one embodiment, the invention includes a method of screeningcompounds for cholesterol modulating activity by contacting test agentswith cells and determining whether the test agents has an effect oncholesterol activity, cholesterol concentration or both in the cellmembrane.

In a further embodiment, determining whether the one or more test agentshas an effect on cholesterol activity, cholesterol concentration or bothin the cell membrane includes contacting the cells with a lytic compoundthat causes perforation or lysis of the membrane of the cells when thecholesterol activity, cholesterol concentration or both of the membraneof the cells reaches a level at or above a threshold cholesterol level.

In yet another embodiment, the cholesterol content of the membrane maybe either increased or decreased by contacting the cells with acholesterol modulating compound. In some embodiments, this will beperformed prior to, simultaneous with or subsequent to contact with thetest agents.

In another embodiment, the invention includes a method of identifying acompound that modulates cholesterol activity by identifying test agentsthat modulate the cholesterol activity in a cell membrane, where thetest agents has been identified by a method in any one of the precedingparagraphs.

In a further embodiment, the invention comprises a method ofmanufacturing a compound that modulates cholesterol activity bysynthesizing or isolating therapeutic agents identified by the methodsof identifying a compound that modulates cholesterol activity.

In yet a further embodiment, the invention includes a method ofmodulating the cholesterol level of a cell by contacting cells with aneffective amount of octanol, ceramide, diglyceride, lysophosphosphatidylcholine, or a combination thereof, thereby increasing or decreasing thecholesterol level of the cells. In certain cases, the cells will be invivo, while in other cases, the cells will be in vitro.

A specific embodiment of the invention includes a kit for determiningthe effect of a test agent on the cholesterol activity, cholesterolconcentration and/or both in a cell membrane. The kit may includes theinstructions for carrying out the method of any one of the previousparagraphs and one or more needed reagents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the screening of lysis of red blood cells usingamphotericin B by turbidity. Human red blood cells were either freshlydrawn or, taken from an outdated blood bank source and then washed andsuspended in physiological buffer. The cholesterol content of washedhuman red blood cells was varied withmethyl-β-cyclodextrin/methyl-β-cyclodextrin-cholesterol complexes. Thecells were distributed into a 96-well plate and incubated for a fewminutes with saline (◯), 0.2 μmol/well octanol (∇) or 1 μg/welllysophosphatidylcholine (□). Amphotericin B (20 μg/well) was added andthe plate incubated for 1 hour at room temperature. As shown in FIG. 1,the variation of endogenous cholesterol content bymethyl-β-cyclodextrin/ methyl-β-cyclodextrin-cholesterol complexes,results in cells that will not be lysed by amphotericin B unless a testagent increases the cholesterol concentration, cholesterol activityand/or both in the cell membrane. In FIG. 1, turbidity (signifyingintact cells) was determined in a plate-reading photometer as opticaldensity (OD) at 500 nm; loss of OD signifies lysis. In FIG. 1, resultsare plotted versus total human red blood cell cholesterol, assessed withAmplex™ Red, Molecular Probes, Eugene, Oreg. The cholesterol content ofunmodified red blood cells was 0.55 μg/μl cells, close to the thresholdof the control curve.

FIG. 2 shows a visual screen of red blood cell lysis caused byamphotericin B. The experiment was as in FIG. 1. Cells with modifiedcholesterol were placed in the conical wells of a 96-well plate, brieflypre-incubated with the two test compounds, and then treated withamphotericin B (20 μg/well) for one hour at room temperature. The platewas photographed after the cells settled. Row A: saline control. Cellsdepleted of cholesterol (wells 1-6) resisted lysis (seen as a loss ofthe central button). Row B: n-octanol (0.2 μmol/well). Even cholesteroldepleted cells were lysed by amphotericin B in the presence of octanolbut not in controls without amphotericin B (not shown). Row C:Lysophosphatidylcholine (1 μg/well). Even enriched cells resistedamphotericin B lysis.

FIG. 3 shows the screening of red blood cell lysis using cholesteroloxidase. The cholesterol content of washed human red cells was variedwith methyl-β-cyclodextrin-cholesterol complexes. The cells weredistributed into a 96-well plate and incubated with saline (◯), 0.2μmol/well octanol (∇) or 1 μg/well lysophosphatidylcholine (□).Cholesterol oxidase (2 IU/ml) was added and the plate incubated for 1 hat 37° C. Turbidity (signifying intact cells) was determined in aplate-reading photometer as O.D. at 500 nm. Loss of turbidity reflectscell lysis.

FIG. 4 depicts the effect of octanol on fibroblast cholesterol.Replicate flasks of fibroblasts were incubated for 24 h in growth mediumwith octanol. The cells were then assayed for cholesterol and protein.

FIG. 5 shows the effect of ceramide and diglyceride on the oxidation ofred cell membrane cholesterol by cholesterol oxidase. The red cellmembranes were pretreated with ceramide or diglycerides and then treatedwith cholesterol oxidase for a hour. An increase in oxidation followingcholesterol oxidase treatment signifies an increase in active cellularcholesterol.

FIG. 6 demonstrates the effect of ceramide and diglyceride on the lysisof red blood cells by saponin. Red blood cells were pre-treated witheither octanol, diglyceride, ceramide, or solvent before incubation withvarying concentrations of saponin. The decrease in optical absorbance at500 nm indicates increasing lysis.

DETAILED DESCRIPTION

Described herein are compounds and methods for modulating cholesterollevels. Also provided are methods for screening compounds, for exampletest agents, for their ability to modulate cholesterol levels, bothsystemically and in cells.

One embodiment described herein provides a method of screening compoundsfor cholesterol modulating activity. Such a method involves exposing acell to a compound, such as a test agent, and measuring the cholesterollevel of the cell or certain cellular components, such as the plasmamembrane, which contains the highest amount of cellular cholesterol, orendoplasmic reticulum, to determine the effect the compound has on thecholesterol levels of the cell or cellular component. Cholesterol levelcan be reflected as cholesterol activity, cholesterol content and/orcholesterol concentration, as desired. As used herein, a test agent is achemical compound that has no known modulating effect on a cell'scholesterol level or cholesterol activity. Accordingly, one skilled inthe art will understand that the term test agent is dependent onmultiple factors including at least the compound to be tested and thecell that is used. The effect the compound has, if any, on thecholesterol activity of the cell can be correlated with cholesterolmodulating properties for the compound.

One method for measuring the cholesterol level of the cell includescontacting the cells with a lytic compound that results in disruption ofcholesterol containing membranes and determining whether the cellmembrane has been disrupted or lysed. In this embodiment, the testcompound can be washed away from, or left in contact with, the cellprior to contact with the lytic agent as desired. The degree of celllysis can also be measured as not all of the cells may lyse. Typically,the lytic compounds will disrupt the cholesterol containing membranewhen the cholesterol level of the membrane is at or above a thresholdlevel, below which little or no cell lysis will occur. As such, lysis ofthe cell membrane provides an indication that the cholesterol level ofthe cell membrane is at or above the threshold level. In someembodiments, the lytic compounds can have a threshold value that liesnear the normal physiological level of the cell used in the assay. Redblood cells typically have a cholesterol content of approximately 0.8moles per mole phospholipids or 0.25 mg cholesterol per mg membraneprotein. Cultured human fibroblasts typically contain approximately 30μg cholesterol per mg cell protein. Factors that might affect thethreshold level of a lytic agent might be the lipid composition of thecells, the type and concentration of drugs added to alter the thresholdand experimental conditions such as incubation temperature and time ofexposure. Examples of lytic compounds that are suitable for use in thepresent methods include lysophosphatides (mono-acyl derivatives ofdiacyl phospholipids), antibiotics, such as polyene antibiotics, andcholesterol oxidase. Exemplary of lysophosphatides is lysophosphatidylcholine. Suitable examples of polyene antibiotics include AmphotericinB, Amphotericin A, Amycin, Ayfactin, Azalomycin F, Candicidin A,Candicidin B, Candimycin, Copiamycin, Filipin, Flavofungin, Fradicin,Hamycin, Lucensomycin, Lucknomycin, Mediocidin, Mepartricin, MepartricinB, Natamycin, Niphimycin, Nystatin Antibiotic Complex, Partricin,Partricin A, Partricin B, Pentamycin, Perimycin A, Proticin, Rimocidin,Sistomycosin, and Sorangicin A. The threshold level for amphotericin Binduced lysis is in the range of the membrane cholesterol level, on amolar basis. Bacterial toxins, particularly those that targetcholesterol or the plasma membrane, may also be used as lytic compounds.Bacterial toxins that may be used as lytic compounds includeperfringolysin O from Clostridium perfringens, hemolysin fromEscherichia coli, listeriolysin O from Listeria monocytogenes, α-toxinfrom Staphyloccocus aureus, pneumolysin from Streptococcus pneumoniae,and streptolysin O from Streptococcus pyogenes. Such bacterial toxinsare discussed in the following: Heuck et al., J. Biol Chem. 2003 Aug.15;278(33):31218-25; Ramachandran et al., Nat Struct Biol. 2002November;9(11):823-7; Schmitt et al., Emerg Infect Dis. 1999March-April;5(2):224-34; and Gilbert R J., Pore-forming toxins. Cell MolLife Sci. 2002 May;59(5):832-44. Sterol-specific lysins, such assaponins may also be used. One skilled in the art will be able toappreciate that any lytic compound which causes cell lysis based oncholesterol level is suitable for use herein, regardless of themechanism of action such lytic compound uses. Depending upon thecholesterol level of the cells used, the lytic agent can be addedbefore, simultaneous with, or subsequent to cell exposure to the testcompound. For example, if the cholesterol level of the cell is below thethreshold value for the lytic agent then the lytic agent can be presentthe entire time whereas if the level is above the threshold value thenthe lytic agent should be added after the cell has been exposed to thecompound being tested. Cell that have cholesterol levels above or belowthe threshold level can be used and will generally depend on whether anincrease or decrease or cholesterol level may be expected.

Regardless of the type of lytic compound, cellular lysis and/orincreased cellular permeability may be measured using a variety ofmethods. In one instance, cell permeability can be measured by a releaseof ions with an ion indicator, such as SBFI (sodium ions), PBFI(potassium ions) and/or Fura-2 (calcium ions) and in particular the cellimpermeable forms of these indicators. See, e.g., Kowalczyk et al. AnalBiochem. 1997 Feb. 1;245(1):28-37. Additionally, cell turbidity can bemeasured, such as with a photometer or by eye. Generally, loss ofturbidity reflects cell lysis. Cell permeability and lysis can also bemeasured using dye exclusion assays, where cells with an intact membraneare able to exclude the dye while cells without an intact membrane takeup the dye. Suitable exclusion dyes include trypan blue, erythrosin andnaphthalene black. A dye uptake stain can be used to measure viabilityas well. In this case, the dye is normally taken up by viable cells butnot by the non-viable cells. Diacetyl fluorescein is an example of a dyeused for dye uptake assays.

Other methods of measuring cell lysis include quantifying release ofradiolabels from cells following cell lysis and/or an increase incellular permeability. Tritium or chromium are commonly usedradiolabels. The release of radiolabels from lysed cells and/or cellswith increased membrane permeability may be measured in various waysincluding scintillation counting and/or film exposure. Additionally,release of measurable enzymes can indicate cell lysis. Enzymes such aslactate dehydrogenase (LDH) are released from cells that have undergonelysis and/or increases in cellular permeability. One way to measure thepresence of lactate dehydrogenase may be to use a colorimetric orfluorometric assay such as CytoTox-ONE™, Promega, Madison, Wis. Fornumerous detailed methods that can be used to measure cell lysis and/orincreases in cellular permeability, see Reed, J. C, Ed., Methods inEnzymology Volume 322: Apoptosis (2000).

Additional methods for measuring cholesterol levels include cellhomogenization or fractionation, such as with a sucrose gradient asdiscussed in Lange et al. J. Lipid Res. 40, 264 (1999) or Chen et al.Gastroenterology. 116(3):678-85 (1999). An example of an approach tomeasuring overall or plasma membrane cholesterol is the use of theenzyme cholesterol oxidase with detection of the product with, forinstance, Amplex™ Red, Molecular Probes, Eugene, Oreg. Total cholesterollevels can also be detected by pelleting and extracting the plasmamembrane in chloroform/methanol and separating the organic extract bythin layer chromatography (TLC). Following separation by TLC,cholesterol may be revealed by spraying the TLC plate with FeCl₃ andheating to 100° C. for 3-5 minutes. Moreover, cholesterol levels may bedetermined using insulin receptor (IR) phosphorylation or cholesterolspecific binding agents, such as filipin, which can be labeled. See,e.g. Mukherjee et al., Biophys J. 1998 October;75(4):1915-25. Moreaccurate and precise are analysis with high-performance liquidchromatography (HPLC) or gas-liquid chromatography (GC).

Types and sources of cells that can be used in the described methods.are not limited as long as the cell contain cholesterol whose level canbe modulated. Accordingly, cells from a wide variety of sources can beused, including both eukaryotic and prokaryotic cells. Typically, thecells will be eukaryotic cells, such as fungal cells or animal cells.When animal cells are used they can be mammalian cells, such as ungulatecells, rodent cells, canine cells, feline cells, porcine cells, and/oraritiodactyla cells. In some embodiments, mammalian cells will beprimate cells, for example human, ape, prosimian and/or monkey cells.Fungal cells used in the screening method may include zygomycetes,ascomycetes, basidiomycetes, and/or mycomycotes.

Suitable types of cells that can be used in the methods, include bloodcells, both red and white, tissue cells, organ cells, skin cells,connective tissue cells, and the like. Typically, cells that have adetermined level of cholesterol in their cellular components will beused to simplify interpretation of the data. In some embodiments, cellswith vigorous cholesterol homeostasis, such as fibroblasts, hepatomacells or macrophages can be used so that a compounds effect oncholesterol homeostasis can be measured. Vigorous cholesterolhomeostasis means prompt and strong responses by the cell to smallvariations in cholesterol; e.g., through cholesterol esterification orbiosynthesis. Cells that are responsible for cholesterol uptake orsynthesis, including liver cells, can also be used.

The cells used in the present assay can also be obtained from a widevariety of sources. For example, cells can be collected freshly from aspecific source and/or stored sources. Cells can also be obtained fromestablished or primary cell lines. In addition, all cells may beisolated from sources containing normal levels of cell membranecholesterol and/or sources containing either abnormally high orabnormally low levels of cell membrane cholesterol. The cells may beplated or free in solution, including cells in media and/or biologicalfluids, either in vivo or in vitro. Screening can be performed inmulti-well plates. Additionally, the present culture system can also beused to mimic different pathological states in cholesterol by usingcells that have defective cholesterol levels or homeostasis. In thisembodiment, as in others, it may be desirable to isolate cells that areknown to have a certain cholesterol defect, such as a genetic defect.

One screening method involves contacting red blood cells with a testagent and measuring the cholesterol level or activity in the plasmamembrane of the red blood cells with a lytic agent, such as amphotericinB, cholesterol oxidase, or saponin. When the cholesterol level oractivity of the red blood cell is above a threshold level, the lyticagent causes disruption of the red blood cell membrane which causes thecells to leak hemoglobin into the surrounding media. This freehemoglobin can be easily detected via a photometer or simply by eyebecause it results in a consistent red solution in which the color doesnot settle out of solution. In contrast, unlysed red cells will settleout of the media and collect in the bottom of the container. As such,some embodiments can use conical shaped micro-well containers so thatthe red cells can settle and collect in a small area to provide a simplevisual indication of cell lysis, for example by producing a central“button.” This screen also provides a simple measure of the degree orpercentage of cell lysis that occurs and provides a simple,straightforward primary method for measuring the effect a compound hason cholesterol levels because the red cells are economical and notcomplex.

Another screening method involves exposing fibroblasts to a testcompounds and measuring the compound's effect on the fibroblast's plasmamembrane cholesterol level with a lytic agent, for example amphotericinB, cholesterol oxidase or saponin. Fibroblasts have vigorous cholesterolhomeostasis and thus provide a good indication of a compound's effect onlive cells. As such, this system can provide an effective secondaryscreening method for following-up on promising compounds. One skilled inthe art that this screening method can also be used as a primaryscreening method, if desired.

In the described methods, cell cholesterol level or activity can beincreased or decreased prior to their use in the screening method. Inthis manner, the cholesterol level or activity of the cells or theircomponents can be adjusted to a desired level. Examples of compoundswhich can decrease endogenous cholesterol levels are cyclodextrins,cyclophanes, phospholipid vesicles, lipid-free/lipid-poorapolipoproteins, reconstituted and native high density lipoprotein(HDL), cholesterol binding compounds, including certain antibiotics suchas filipin, nystatin, digitonin, and/or streptolysin, and whole serum.As will be apparent to the skilled artisan, when antibiotics are used toreduce cholesterol levels antibiotics or amounts that result in celllysis should not be used. Compounds that can be used to increasecholesterol levels include cyclodextrin-cholesterol complexes. Suitablecyclodextrins include beta-cyclodextrins and their derivatives, such asmethyl-beta-cyclodextrin or hydroxy-propyl-β-cyclodextrin. Suchcompounds and techniques are discussed in Klein et al. Biochemistry.1995 Oct. 24;34(42):13784-93, Christian et al. J Lipid Res. 1997November;38(11):2264-72, Christian et al. J Lipid Res. 1999August;40(8):1475-82 and Rothblat et al. J Lipid Res. 1999May;40(5):781-96. Cellular cholesterol may also be depleted by culturein a media devoid of exogeneous cholesterol sources. Additionally,cholesterol biosynthesis may be inhibited by cholesterol inhibitingcompounds. One group of these cholesterol inhibiting compounds includesthe statins. Specifically, statins such as compactin, lovastatin and/orsqualestatin block cholesterol production. Accordingly, cell cholesterolcan be adjusted to a broad range of values, including increments of 10%,20%, 25%, 30%, 50%, 75%, or more, up to two-fold or more.

These methods can further involve repeating the screening method one ormore times, simultaneously, e.g. in a multi-well or parallel format, orsubsequent to one another. In the repeats, one or more of the differentparameters of the screening method, e.g. test compound, cell type,cholesterol level, cholesterol level measurement, etc., can be varied asdesired. For example, an array of screens can be performed on differentcompounds simultaneously keeping all other conditions the same using amulti-well format. In this manner a large number of compounds, such as5, 10, 25, 50, 100 or more, can be simultaneously screened for theircholesterol level modulating properties. In these and other formats, thesame compound can be tested on different types of cells and/or on thesame cell type pre-treated in different ways. One or more compounds canalso be individually tested against a panel of two, three, four, five,six, seven, eight, nine, ten or more samples of the same type of cellsthat have different cholesterol levels, which levels can be staggered atregular or irregular intervals. These varied cholesterol levels can beachieved as described herein. Typically, cells of the same types used inthe present methods will come from a single source and have similar orsubstantially homogeneous properties in order to simplify interpretationof assay results. Although such panel and array formats typically willuse a multi-well format for simplicity, different containers can also beused for each sample.

The invention can be adapted for either low or high throughput screeningof the effect of a test agent on the membrane cholesterol concentration,cholesterol activity or both of one or more cells. Cholesterol activitymeans the state of cholesterol beyond that constrained by membranephospholipids such that it is more or less reactive with probes, such ascholesterol oxidase and cyclodextrins, and more or less capable of goingto the endoplasmic reticulum and increasing or decreasing itscholesterol. Typically cholesterol activity is independent ofcholesterol concentration in the membrane. Chemical activity ofcholesterol is also discussed by Radhakrishnan et al., Proc Natl AcadSci USA 2000 Nov. 7; 97(23):12422, and in other references cited herein.The screening method encompasses a method that is suitable, and istypically used, for assaying for a particular property or effect in alarge number of test agents. However, individual test agents may beused. Typically, the screening method requires only a small amount oftime for each test agent assayed; characteristically more than one testagent is assayed simultaneously (as in a 96-well microtiter plate), andpreferably significant portions of the procedure can be automated. Infact, the present methods easily lend themselves to automation.

As described herein the effect of the test compound may be measured byany number of methods. In order to provide a gauge against which tomeasure the effect of the test agent, the results achieved with thecompounds can be compared to a control, such control may include anyassay done using the same experimental conditions but without theaddition of the test agent or by comparing the results of the effect ofthe test agent with the total amount of cellular cholesterol.Preferentially, the methods used to measure the effect of a test agentinclude cell lysis and/or an increase in cell permeability caused by alytic compound. However, other measures of cholesterol activity such asits transfer to cyclodextrin or changes in endoplasmic reticulumcholesterol can be used.

Typically, the present methods focus on cholesterol levels in the plasmamembrane of cells because the vast bulk of the cell's cholesterol isassociated with the plasma membrane. However, the homeostatic effectorsthat set the level of this cholesterol reside mostly in the endoplasmicreticulum (ER) and small variations in plasma membrane cholesterol nearits physiological level can rapidly change ER cholesterol by a factor of20 or more (FIG. 1). Without limiting the scope of the invention, it isbelieved that for regulatory purposes, the level of cholesterol in theplasma membrane sets the abundance of cholesterol in the ER which thenmakes the adjustments on behalf of the plasma membrane and the cell as awhole. In particular, a diverse set of regulatory mechanisms in the ERtranslate fluctuations in its cholesterol into homeostatic responses,restoring the plasma membrane to normal. The examples described belowevidence that this homeostatic system not only responds to the level ofplasma membrane cholesterol itself but also to alterations in theproperties of the plasma membrane lipid bilayer. Thus, it is believedthat the mechanism underlying this behavior is that the signal to the ERreflects the activity of the cholesterol in the plasma membrane ratherthan its absolute concentration. Radhakrishnan et al. Biochemistry. 2000Jul. 18;39(28):8119-24. The downstream consequences of a rise in plasmamembrane cholesterol activity can result in an increase in the ERcholesterol pool size leading to a decrease in cell cholesterol minutesor hours later. In contrast, a fall in plasma membrane cholesterolactivity can have the opposite effect. Accordingly, the present assaymay identify compounds that modulate cell cholesterol levels by actingon the cholesterol activity of the plasma membrane and/or ER. In someembodiments, such compounds may raise the activity of the cholesterol inthe plasma membrane, perhaps lodging in the plasma membrane bilayer,cause an increase in ER cholesterol, and thereby signal the cells todecrease cholesterol accumulation. Drugs of this type would reduce totalbody cholesterol by an entirely new mechanism. Additionally, becausesuch drugs do not block cholesterol precursor molecules needed in thesynthesis of other important cellular constituents, such as happens withstatins, they will likely cause fewer side effects.

After a compound or test agent is identified as having a desiredproperty, such as lowering cholesterol amounts, the test agent can beidentified and then isolated, provided or chemically synthesized toproduce a therapeutic drug. Thus, the present methods can be used tomake drug products that modulate cellular cholesterol production and areuseful for the therapeutic treatment of cholesterol disorders in vitroand in vivo. In some embodiments, such compounds will increase thecholesterol activity of a cell's plasma membrane or ER while in othersthe compounds will decrease the cholesterol activity of a cell's plasmamembrane or ER. Such compounds can be used to treat cholesterolassociated diseases and disorders, such as atherosclerosis andhypercholesterolemia. When used to treat such disorders the compoundswill typically be provided or administered as a pharmaceuticallyacceptable composition.

The described screening methods have been used to identify compoundswhich modulate cholesterol activity, which can either upregulate ordownregulate cholesterol production. As can be seen from the examples,octanol increases plasma membrane cholesterol activity in both red cellsand human fibroblasts. In fact, in human fibroblasts, prolonged culturewith octanol resulted in cholesterol reduction. The examples alsodemonstrate that in red blood cells similar effects to octanol are seenwith physiologic membrane-intercalating alcohols such as ceramides anddyglycerides. Other fatty alcohols likely behave similarly, and adiverse class of such agents might be discovered by the assays describedherein.

In contrast, the described screening methods identified thatlysophosphatidylcholine has the opposite effect of octanol, ceramides,and diglycerides on certain cells and decreases plasma membranecholesterol levels or activity which may result in increased cellcholesterol content. A diverse class of such agents might be discoveredby the assays described herein.

Compounds that increase plasma membrane cholesterol activity, in themanner of octanol, ceramides and dyglycerides might also find use as anadjunct to the treatment of systemic fungal infections with polyeneantibiotics such as amphotericin B, since a small reduction in thecholesterol content of the plasma membrane would protect the body cellsfrom the toxicity of these sterol directed agents. Norman et al. Adv.Lipid Res., 14, 127-70. Compounds that can increase the sterol(typically, ergosterol) level of fungal cells, such as through selectivetargeting or fungal cell versus human cell specificity, may be used tomake the fungal cells more susceptible to polyene antibiotic treatment.Similarly, individuals afflicted with bacterial infections producingsterol-directed toxins (e.g., streptolysin-d) might benefit from drugsthat lower their plasma membrane cholesterol below the thresholdrequired for the action of these toxins. Duncan et al. Biochim BiophysActa, 603, 278-87.

The present invention also provides kits for carrying out the methodsdescribed. In one embodiment, the kit is made up of instructions forcarrying out any of the depicted methods. The instructions can beprovided in any intelligible form through a tangible medium, such asprinted on paper, computer readable media, or the like. The present kitscan also include one or more reagents, cells, buffers, culture media,culture media supplements, lytic compounds capable of causing lysis ofone or more cells, specific cholesterol binding compound(s) for labelingcholesterol, chromatic or fluorescent dyes for staining or labeling thespecific cholesterol binding compound(s), radioactive isotopes, and/ordisposable lab equipment, such as multi-well plates. Examples ofsuitable kit components are described herein and set forth in theexamples below. The main purpose of these kits is to readily facilitateimplementation of the present methods.

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Screen Using Red Blood Cell Lysis by Amphotericin B

Human red cells (freshly drawn or from an outdated blood bank source)were washed and suspended in physiological buffer. The red cell'scholesterol content was lowered slightly (e.g., by 20%) withcyclodextrin (Steck et al., Biophys. J., 83, 2118-25, 2002) so that theywould not be lysed by amphotericin B unless a favorable compoundincreased the activity of the cholesterol in their membrane. Thecholesterol modified cells were placed in the wells of a microtiterplate and incubated for a few minutes at room temperature with octanoland lysophosphatidylcholine, which can be replaced with a battery ofdrugs (individually or in mixtures). Amphotericin B was then added andthe plate incubated at room temperature for an hour. Cell lysis wasscored by reading light scattering in a plate-reading photometer(FIG. 1) or, more simply, by letting the unlysed cells settle for anhour in conical wells and then assessing hemolysis by eye (FIG. 2). Asseen in FIGS. 1 and 2, octanol favors amphotericin B lysis, suggestingthat it increases the activity of membrane cholesterol whilelysophosphatidylcholine has the opposite effect.

Discussion of Results

Polyene antibiotics such as amphotericin B are known to promote thedisruption of cholesterol containing membranes. However, this actiondepends critically on the cholesterol level in the membrane (see thecontrol curves, O---O, in FIGS. 1 and 2). The critical cholesterolcontent for lysis is close to the physiological level in humanfibroblast plasma membranes, for the same reason that ER cholesterolresponds dramatically near the physiological content of the plasmamembrane. Without limiting the scope of the invention it is believedthat the threshold observed in these assays reflects the saturationpoint of the membrane lipid bilayer and that cholesterol in excess ofthis point has a high activity and behaves in a special fashion. Drugsthat raise the activity of plasma membrane cholesterol can therefore berecognized by their ability to increase cell susceptibility toamphotericin B lysis. Drugs of interest are those which potentiate celllysis; presumably, by increasing the activity of plasma membranecholesterol. Octanol is such a compound (∇---∇ in FIGS. 1 and 2).Conversely, agents that reduce cholesterol activity will protect againstcell lysis; an example of which is lysophosphatidyl choline (◯---◯ inFIGS. 1 and 2). Accordingly, this assay will be used to screen chemicalsfor their ability to raise the activity of plasma membrane cholesterolwhich should reduce the production of cholesterol in vivo.

Example 2 Screen Using Red Blood Cell Lysis with Cholesterol Oxidase

The procedure used in this Example were the same as those for Example 1,except that cholesterol oxidase was substituted for amphotericin B. Thisexample demonstrates that cholesterol oxidase also reports on membranecholesterol activity. As with amphotericin B, the cholesterol oxidaseenzyme scarcely acts upon cholesterol below a threshold that lies nearthe physiological level but vigorously oxidizes cholesterol once thatthreshold is reached. Lange et al. Biochim Biophys Acta, 769, 551-62.The oxidation of red cell membrane cholesterol leads to lysis. Promisingagents can therefore be readily detected in a multiwell screen similarto that described above in Example 1. The results of this experiment areillustrated in FIG. 3.

Example 3 Screen Using Fibroblast Cell Lysis Detected with CholesterolOxidase

A human fibroblast cell line is grown in bulk culture and seeded intomicrotiter wells the day before experimentation. The fibroblast cellsfrom the fibroblast cell line are rinsed and treated with octanol pluscholesterol oxidase as in Example 2. The increased level of plasmamembrane cholesterol activity is detected in two ways. First, plasmamembrane cholesterol activity is detected by susceptibility tocholesterol oxidase. Although cholesterol in human fibroblast is a poorsubstrate for cholesterol oxidase under standard conditions, it isreadily oxidized when the plasma membrane cholesterol activity isincreased; for example, by raising its concentration by 20-55% or byadding 0.1-1.0 mM octanol. Cell lysis caused by cholesterol oxidationcan also be determined by measuring increased cell permeability using aplate fluorescence reader and the impermeable indicator of potassiumions, PBFI, or another indicator system.

Example 4 Assessment of Drug Action Using Fibroblasts and Amphotericin B

The cultured cells described in Example 3 will be plated in microtiterwells and incubated with test compounds at 37° C. for two days to allowa full metabolic response. The drug will then be washed away,amphotericin B added to the wells and cell lysis followed from theleakage of potassium ions or another indicator, as above. Resistance toamphotericin B lysis will indicate that the objective has been achieved:the reduction of plasma membrane cholesterol in response to the testcompound. Other polyene antibiotics (e.g., nystatin and filipin) will betested in place of amphotericin B in order to discern their effects. Incontrast to the screens described in the previous examples, which areused to identify compounds that raise the activity of membranecholesterol, the present assay will measure the long-term effect ofmodulating cholesterol levels. Agents that increase plasma membranecholesterol activity, when used as drugs, will signal cells to reducetheir cholesterol accretion homeostatically. The reaction of fibroblastswith amphotericin B can then be used to detect the response of livingcells to such drugs; namely, the homeostatic reduction of plasmamembrane cholesterol. This is because agents that immediately raise theactivity of plasma membrane cholesterol should, over time, lead to thereduction of the cholesterol content of the plasma membranes of livingcells. This can be detected by their increased resistance toamphotericin B, which normally lyses these cells. The results for thisexperiment using octanol on fibroblasts is shown in FIG. 4.

Example 5 Identifying Drugs by Screen Using Red Cell Membrane LysisDetected with Cholesterol Oxidase

Red cell membrane lysis is measured using cholesterol oxidase as setforth in the examples above. Prior to lysis, red cell membranes aretreated with either differing amounts of a ceramide analog (0-12 μmolar)or differing amounts of a diglyceride analog (0-12 μmolar). Theincreased level of plasma membrane cholesterol activity is detected bysusceptibility to cholesterol oxidase and measured as a % of oxidation.As demonstrated in FIG. 5, both ceramide and diglyceride increase thesusceptibility of red cell cholesterol to oxidation.

Example 6 Drugs Identified by Screen Increase Lysis of Red Blood Cellswith Saponin

Red cell membranes are treated with octanol, diglyceride, ceramide, orsolvent before incubation with different concentrations of saponin (0-8μg/well) for 30 minutes. Optical absorbance at OD₅₀₀ is measured.Decreased optical absorbance indicates increasing lysis by saponin. Asdemonstrated by FIG. 6, octanol (∇), diglyceride (▪), and ceramide (⋄)all increase lysis of red blood cells at lower concentrations of saponinas compared to control (♦).

Example 7 Follow-up Testing

The properties of agents found to be desirable in these screens will beresearched in the literature for possible serious adverse effects oncells or organisms in vitro or in vivo. In addition, these compoundsshould also be potent enough in vitro to suggest the possibility oftherapeutic use in humans at a tolerable dose (e.g., less than one gramor 10 mg/kg body weight per day). The agents should be absorbable fromthe gastro-intestinal tract. Selected compounds will be further tested.In an initial simple characterization, agents of interest should showthe following desired properties when applied to a variety of cell typesgrown in standard culture: reduced total cell and plasma membranecholesterol, measured per cell, per unit protein or per phospholipid.Proof of principle is demonstrated for octanol in FIG. 4 and ceramideand diglyceride in FIG. 5 and FIG. 6. Biochemical tests will be used toverify that treated cells indeed incorrectly sense that they have excesscholesterol on board. For example, they should reduce their HMG-CoAreductase and LDL receptor levels. Treated cells should have near normalgrowth, as measured by the fractional rate of protein accretion. Thereshould be near normal cell morphology and ultrastructural organization.The profile of other cell membrane lipids should also be essentiallyunchanged. Other evidence of cell injury (e.g., apoptosis) should beabsent. Procedures: established methods will be used for theaforementioned measurements. See Reed, J. C, Ed., Methods in EnzymologyVolume 322: Apoptosis (2000).

The present methods can involve any or all of the steps or conditionsdiscussed above in various combinations, as desired. Accordingly, itwill be readily apparent to the skilled artisan that in some of thedisclosed methods certain steps can be deleted or additional stepsperformed without affecting the viability of the methods. Furthermore,as used herein, including specifically the language in the specificationand the claims, use of “a” or “an” means “one” or “one or more.”

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges. Any listed range can be easily recognizedas sufficiently describing and enabling the same range when broken downinto at least equal halves, thirds, quarters, fifths, tenths, etc. As anon-limiting example, each range discussed herein can be readily brokendown into a lower third, middle third and upper third, etc. As will alsobe understood by one skilled in the art, all language such as “up to,”“at least,” “greater than,” “less than,” “more than” and the likeinclude the number recited and refer to ranges that can be subsequentlybroken down into subranges as discussed above. In the same manner, allratios disclosed herein also include all subratios falling within thebroader ratio.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, thepresent invention encompasses not only the entire group listed as awhole, but each member of the group individually and all possiblesubgroups of the main group. Accordingly, for all purposes, the presentinvention encompasses not only the main group, but also the main groupabsent one or more of the group members. The present invention alsoenvisages the explicit exclusion of one or more of any of the groupmembers in the claimed invention.

All references disclosed herein are specifically incorporated byreference thereto. The following references are specificallyincorporated.

1. Simons K, Ikonen E. (2000) How cells handle cholesterol. Science,290, 1721-6.

2. Lange Y, Steck T L. (1996) The role of intracellular cholesteroltransport in cholesterol homeostasis. Trends in Cell Biology, 6, 205-08.

3. Lange Y, Ye J, Rigney M, Steck T L. (1999) Regulation of endoplasmicreticulum cholesterol by plasma membrane cholesterol. J. Lipid Res., 40,2264-70.

4. Radhakrishnan A, McConnell H M. (2000) Chemical activity ofcholesterol in membranes. Biochemistry, 39, 8119-24.

5. Norman A W, Spielvogel A M, Wong R G. (1976) Polyeneantibiotic-sterol interaction. Adv. Lipid Res., 14, 127-70.

6. Duncan J L, Buckingham L. (1980) Resistance to streptolysin O inmammalian cells treated with oxygenated derivatives of cholesterol.Cholesterol content of resistant cells and recovery of streptolysin Osensitivity. Biochim Biophys Acta, 603, 278-87.

7. Steck T L, Ye J, Lange Y. (2002) Probing red cell membranecholesterol movement with cyclodextrin. Biophys. J., 83, 2118-25.

8. Lange Y, Matthies H, Steck T L. (1984) Cholesterol oxidasesusceptibility of the red cell membrane. Biochim Biophys Acta, 769,551-62.

9. Lange Y, Ye J, Steck T L. (2004) How plasma membrane cholesterol inexcess of phospholipids regulates cholesterol homeostasis. Proc. Natl.Acad. USA, 101, 11664-67.

While preferred embodiments have been illustrated and described, itshould be understood that changes and modifications can be made thereinin accordance with ordinary skill in the art without departing from theinvention in its broader aspects as defined in the following claims.

1. A method of screening compounds for cholesterol modulating activity,comprising: (a) contacting one or more test agents with one or morecells; and (b) determining whether the one or more test agents has aneffect on cholesterol activity, cholesterol concentration or both in amembrane of the one or more cells.
 2. The method of claim 1, wherein (b)comprises contacting the one or more cells with a lytic compound,wherein the lytic compound causes perforation or lyses of the membraneof the one or more cells when the cholesterol activity, cholesterolconcentration or both of the membrane of the one or more cells reaches alevel at or above a threshold cholesterol level.
 3. The method of claim1, further comprising performing (a) and (b) one or more times withdifferent test agents.
 4. The method of claim 3, wherein the differenttest agents are screened simultaneously.
 5. The method of claim 1,further comprising: (c) increasing or decreasing the cholesterol contentof the membrane by contacting the one or more cells with a cholesterolmodulating compound.
 6. The method of claim 5 wherein (c) is performedprior to (a), simultaneous with (a) or subsequent to (a).
 7. The methodof claim 5, wherein the cholesterol modulating compound comprises acyclodextrin or cyclodextrin derivative.
 8. The method of claim 2,wherein the lytic compound comprises a polyene antibiotic.
 9. The methodof claim 2, wherein the lytic compound comprises a lysophosphatide orcholesterol oxidase.
 10. The method of claim 2, wherein the lyticcompound comprises a bacterial toxin.
 11. The method of claim 1, whereinthe one or more cells comprise one or more eukaryotic cells.
 12. Themethod of claim 11, wherein the one or more eukaryotic cells compriseone or more mammalian cells.
 13. The method of claim 11, wherein the oneor more cells comprise one or more red blood cells.
 14. The method ofclaim 11, wherein the one or more cells comprise one or morefibroblasts.
 15. The method of claim 11, wherein the one or more cellscomprise one or more human cells.
 16. The method of claim 1, wherein theone or more cells have vigorous cholesterol homeostasis.
 17. The methodof claim 1, wherein (b) comprises measuring the effect, if any, the testagent has on the cholesterol activity, cholesterol concentration or bothin the membrane of the one or more cells.
 18. The method of claim 1,wherein (b) comprises measuring the permeability of the membrane of theone or more cells or the turbidity of the one or more cells.
 19. Themethod of claim 1, wherein the one or more cells are in vitro.
 20. Themethod of claim 1, wherein the one or more cells are in vivo.
 21. Themethod of claim 1, wherein (b) comprises measuring the cholesterolactivity, cholesterol concentration or both in a plasma membrane of theone or more cells. 22-25. (canceled)
 26. A method of modulating acholesterol level of a cell comprising contacting one or more cells withan effective amount of octanol, ceramide, diglyceride,lysophosphosphatidyl choline, or combinations thereof, therebyincreasing or decreasing the cholesterol level of the one or more cells.27. The method of claim 26 wherein the one or more cells are in vivo.28. (canceled)